effects of gamma-sterilization on physicochemical properties and stability of Nigella sativa OIL and gentamicin formulation

by

nor liyana binti jaafar

international islamic universitymalaysia

2015

viieffects of gamma-sterilization on physicochemical properties and stability of Nigella sativa OIL and gentamicin formulation

By

Nor liyana binti jaafar

A thesis submitted in partial fulfilment of the requirement for the degree of Bachelor of Biomedical Science

Kulliyyah of Allied Health ScienceInternational Islamic University Malaysia

may 2015AbstractSterilization of medical products for systemic application is mandatory before the products can be marketed. Gamma irradiation is one of the widely used method for sterilization. This study aims to determine the effects of gamma-sterilization on the stability and physicochemical properties of Nigella sativa-gentamicin emulsion. Four emulsions (A, B, C and D) with different concentration of N. sativa oil in each samples had been formulated, whereas the concentration of gentamicin was made constant at 0.1% (w/v) in all samples. All of the samples were stored at three different storage conditions (8C, 25C and 50C). 120 mL from each samples were sent for gamma-sterilization. Several stability tests were performed on the samples before and after gamma-sterilization. The stability evaluations include organoleptic characteristics, centrifugation test (5000rpm, 5 minutes), freeze-thaw cycle, pH determination, particle size measurement and zeta-potential analysis. The emulsions of both pre- and post-gamma sterilization had been tested on biofilm-producing bacteria of S. aureus, P. aeroginosa and S. epidermidis. Results showed no colour changes and phase separation were presented to all of the samples that kept at 8C at all-time points. At 25C storage conditions, phase separation was formed at day 14 in all four formulations for both pre- and post-gamma-sterilization samples. Samples kept 50C were the most unstable since colour changes and phase separation formed at day 7 onwards. The particle size of the samples showed significant difference between pre- and post-gamma sterilization. Whereas, no significant different in term of zeta-potential before and after gamma-sterilization. Results showed that samples kept at 8C demonstrated to be the most stable throughout the study.

AbstrakPensterilan produk perubatan untuk aplikasi sistemik adalah wajib sebelum produk boleh dipasarkan. Sinaran gamma adalah salah satu kaedah yang digunakan secara meluas untuk tujuan pensterilan. Kajian ini bertujuan untuk menentukan kesan gamma-pensterilan pada kestabilan dan sifat fizikokimia emulsi Nigella sativa-gentamicin. Empat emulsi (A, B, C dan D) dengan kepekatan minyak N. sativa yang berbeza dalam setiap sampel telah dirumuskan, manakala kepekatan gentamicin dibuat tetap pada 0.1% (w / v) dalam semua sampel. Semua sampel disimpan pada tiga keadaan penyimpanan yang berbeza (8C, 25C dan 50C). 120 mL daripada setiap sampel telah dihantar untuk gamma-pensterilan. Beberapa ujian kestabilan telah dilakukan ke atas sampel sebelum dan selepas gamma-pensterilan. Penilaian kestabilan termasuk ciri-ciri organoleptik, ujian centrifugasi (5000 rpm, 5 minit), kitaran beku-cair, penentuan pH, pengukuran saiz zarah dan analisis zeta-berpotensi. Emulsi untuk sebelum dan selepas gamma-pensterilan telah diuji ke atas bakteria yang menghasilkan biofilem iaitu S. aureus, P. aeroginosa dan S. epidermidis. Keputusan kajian menunjukkan tiada perubahan warna dan pemisahan fasa yang berlaku kepada semua sampel yang disimpan pada 8C. Pada keadaan penyimpanan 25C, pemisahan fasa telah berlaku pada hari ke-14 dalam keempat-empat formulasi untuk pra- dan pasca-gamma-pensterilan. Sampel yang disimpan pada suhu 50C adalah yang paling tidak stabil kerana perubahan warna dan pemisahan fasa terbentuk pada hari ke-7 dan seterusnya. Saiz zarah menunjukkan perbezaan yang signifikan antara sampel pra dan pasca-gamma pensterilan. Sebaliknya, tidak ada perbezaan yang signifikan dari segi zeta-potensi sebelum dan selepas gamma-pensterilan. Hasil kajian menunjukkan bahawa sampel-sampel yang disimpan pada suhu 8C menunjukkan tahap kestabilan yang paling baik sepanjang eksperimen ini berjalan.

Approval pageI certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Bachelor of Biomedical Science

..Asst. Prof. Dr. Mohd Affendi Bin. Mohd ShafriSupervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Bachelor of Biomedical Science

..Name: Examiner 1

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Bachelor of Biomedical Science

..Name: Examiner 2

This thesis was submitted to the Department of Biomedical Science and is accepted as a partial fulfilment of the requirements for the degree of Bachelor of Biomedical Science

..Asst. Prof. Dr. Ibrahim Adham Bin. TaibHead, Department of Biomedical Science

DeclarationI hereby declare that this thesis is the result of my own investigation, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Nor Liyana Binti Jaafar

Signature.Date ..................

v

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIADECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCHCopyright 2015 by International Islamic University Malaysia. All rights reserved.EFFECTS OF STERILIZATION ON PHYSICOCHEMICAL PROPERTIES AND STABILITY OF Nigella sativa AND GENTAMICIN FORMULATION

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries. Affirmed by

Nor Liyana Binti Jaafar

......SignatureDate

vi

AcknowledgementIn The Name of Allah, the Most Gracious, the Most MercifulAlhamdulillah, praised to Allah T for His blessing and mercy, this thesis is completed with given strength and wisdom granted by Him. First and foremost, I would like to express my heartfelt gratitude to my supervisor Asst. Prof. Dr. Mohd Affendi b. Mohd Shafri for his constant guidance and valuable advice throughout my project. His enthusiasm in scientific research really inspired me in completing this study.A million thanks to postgraduate students Sr. Fathin Athirah Yusof and Br Khairul Ikhwan Yaakob for their constant source of encouragement, endless support and valuable advice. Their willingness to help me has motivated me wholly throughout my project. I really do appreciate all their willingness to guide me in order to let me understand, manage the lab operation systems and accomplish the understanding of the project. Besides, I would like express my appreciation to all lab technicians and science officer for their kindness and help in providing me with a good environment and facilities to complete this study.Finally, an honorable mention goes to my families and friends for their boundless understandings and supports. Without helps of the particular that mentioned above, I would face many difficulties in completing my final year project. Last but not least, I would like to thank all those who have involved directly and indirectly throughout my final year project. May the blessing of Allah be upon them.

Table of ContentsAbstractiiAbstract in MalayiiApproval pageivDeclarationvCopyright PageviAcknowledgementviiTable of ContentsviiiList of TablesxList of FiguresxiiList of Symbols / Equationsxiv

Chapter 1: Introduction11.1 RESEARCH BACKGROUND11.2 AIMS AND OBJECTIVES..31.3 HYPOTHESIS..3

Chapter 2: Literature Review42.1 LITERATURE REVIEW.42.1.1 Definition of Sterilization......42.1.2 Advantages of Gamma-Sterilization..52.1.3 Disadvantages of Gamma-Sterilization.62.1.4 Emulsion...72.1.5 Stability Testing of Emulsion....9

Chapter 3: Methodology123.1MATERIALS...123.1.1 Apparatus and Equipment133.1.2 Chemicals and Reagents...133.1.3 Glassware and Disposable/Consumable Items.133.1.4 Microorganisms....143.2 METHODS..143.2.1 Emulsification Process..143.2.2 Stability Tests...163.2.2.1 Organoleptic Characteristics..163.2.2.2 Centrifugation Test....163.2.2.3 Freeze-Thaw Cycle ...163.2.2.4 pH Determination ..173.2.2.5 Particle Size Measurement.173.2.2.6 Zeta Potential Analysis .173.2.3 Gamma-sterilization.183.2.4 Antimicrobial Susceptibility Assays183.2.4.1 Preparation of Disc... 183.2.4.2 Preparation of Inoculum ...183.3.4.3 Disc Diffusion Assay 183.2.5 Statistical Analysis.. .19

Chapter 4: Results and Findings 204.1 RESULTS...214.1.1 Organoleptic Characteristics224.1.2 Centrifugation Test..214.1.3 Particle Size Measurement..244.1.4 Zeta-Potential Analysis264.1.5 pH Measurement 284.1.6 Freeze-Thaw Cycle Test..304.1.7 Antimicrobial Susceptibility Assay.31

Chapter 5: Discussion AND CONCLUSION355.1 DISCUSSION.355.1.1 Introduction..365.1.2 Organoleptic Characteristics345.1.2.1 Color.355.1.2.2 Phase Separation...375.1.3 Centrifugation Test..385.1.4 Particle Size Measurement..385.1.5 Zeta Potential Analysis....395.1.6 pH Measurement..405.1.7 Freeze-Thaw Cycle Test..425.1.8 Antimicrobial Susceptibility Assay.425.2 LIMITATIONS..455.2 CONCLUSION..455.3 FUTURE WORK...47

References48

Appendix53

List of TablesTable No.Page No.

Table 3.1Apparatus and equipment list with model type and brand/ manufacturer

24

Table 3.2List of chemicals and reagents with brand/ manufacture

25

Table 3.3Glassware and disposable/consumable items with brand/manufacturer

25

Table 3.4The formulation of emulsion A, B, C and D with different concentration of Nigella sativa oil and constant concentration of gentamicin

27

Table 4.1Organoleptic characteristics of emulsion a for pre-gamma and post-gamma sterilization kept in three different storage temperature

32

Table 4.2Organoleptic characteristics of emulsion b for pre-gamma and post-gamma sterilization kept in three different storage temperature

32

Table 4.3

Organoleptic characteristics of emulsion c for pre-gamma and post-gamma sterilization kept in three different storage temperature

33

Table 4.4

Organoleptic characteristics of emulsion d for pre-gamma and post-gamma sterilization kept in three different storage temperature

33

Table 4.5

Centrifugation result on the emulsion A of pre- and post-gamma- sterilization kept in three different storage conditions.

34

Table 4.6

Centrifugation result on the emulsion B of pre- and post-gamma- sterilization kept in three different storage conditions

34

Table 4.7

Centrifugation result on the emulsion C of pre- and post-gamma- sterilization kept in three different storage conditions34

Table 4.8

Centrifugation result on the emulsion D of pre- and post-gamma- sterilization kept in three different storage conditions. 35

Table 4.9

Freeze-thaw cycle results of gentamicin-N.sativa emulsion (A, B, C and D) for three cycles before and after gamma-sterilization

42

List of FiguresFigure No.Page No.

Figure 2.1Breakdown process of the emulsion (Adamczyk, 2013)24

Figure 3.1 Schematic diagram of gentamicin-N.sativa emulsion formulation (Emulsion A, B, C and D). The diagram was taken from Fathin et al., (2014).28

Figure 3.2Summary of the methods32

Figure 4.1Mean particle size of emulsion A for pre and post-gamma sterilization kept in variable storage temperature36

Figure 4.2

Mean particle size of emulsion B for pre and post-gamma sterilization kept in variable storage temperature37

Figure 4.3

Mean particle size of emulsion C for pre and post-gamma sterilization kept in variable storage temperature37

Figure 4.4

Mean particle size of emulsion D for pre and post-gamma sterilization kept in variable storage temperature38

Figure 4.5Mean zeta-potential of emulsion A for pre and post-gamma sterilization kept in variable storage temperature38

Figure 4.6

Mean zeta-potential of emulsion B for pre and post-gamma sterilization kept in variable storage temperature39

Figure 4.7Mean zeta-potential of emulsion C for pre and post-gamma sterilization kept in variable storage temperature39

Figure 4.8

Mean zeta-potential of emulsion D for pre and post-gamma sterilization kept in variable storage temperature40

Figure 4.9

Mean pH value of emulsion A for pre and post-gamma sterilization kept in variable storage temperature40

Figure 4.10Mean pH value of emulsion B for pre and post-gamma sterilization kept in variable storage temperature41

Figure 4.11

Mean pH value of emulsion C for pre and post-gamma sterilization kept in variable storage temperature41

Figure 4.12

Mean pH value of emulsion D for pre and post-gamma sterilization kept in variable storage temperature42

Figure 4.13The mean of inhibition zone of P. aeroginosa OS00443

Figure 4.14The mean of inhibition zone of P. aeroginosa ATCC 44

Figure 4.15The mean of inhibition zone S. aureus OS00144

Figure 4.16The mean of inhibition zone S. aureus OS00345

Figure 4.17The mean of inhibition zone S. aureus ATCC45

Figure 4.18The mean of inhibition zone of S. epidermidis OS01 (Resistant group)46

Figure 4.19The mean of inhibition zone of S.epidermidis ATCC (Control group46

List of Symbols / ABBREVIATIONSymbols/ Abbreviation

O/W

Oil-in-Water

W/O Water-in-Oil

F/T Freeze-Thaw

nm

nanometer

C degrees of Celcius

rpm

Revolutions per minute

NSO

Nigella sativa oil

mV

millivolt

cfu

colony forming unit

v/vvolume per volume

x